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Abstract:

Peptide compositions comprising a dimeric peptide which combines two
peptide monomers, each independently comprising the amino acid sequence:
Asp-X1-X2-Asn-Tyr-Ile-Thr-X3
wherein: X1 is selected from the group consisting of Cys and a Cys
derivative; X2 is selected from the group consisting of Cys and a Cys
derivative; and X3 is selected from the group consisting of Arg and
Glu-Leu-Arg, provided that at least one of X1 and X2 is Cys, whereby a
mol percentage of the dimeric peptide in the composition is at least 50
mol percents, or at least 99 mol percents, are disclosed. Further
disclosed are processes of preparing such peptide compositions and uses
thereof in the treatment of allergic disorders.

Claims:

1-40. (canceled)

41. A peptide composition comprising a dimeric peptide, said dimeric
peptide comprising two peptide monomers, each independently comprising
the amino acid sequence: Asp-X1-X2-Asn-Tyr-Ile-Thr-X3 wherein: X1 is
selected from the group consisting of Cys and a Cys derivative; X2 is
selected from the group consisting of Cys and a Cys derivative; and X3 is
selected from the group consisting of Arg and Glu-Leu-Arg, provided that
at least one of X1 and X2 is Cys, wherein a mol percentage of said
dimeric peptide in the composition is at least 50 mol percents.

42. The peptide composition of claim 41, wherein at least one of said
peptide monomers comprises said amino acid sequence in which one of X1
and X2 is said Cys derivative.

43. The peptide composition of claim 41, wherein said Cys derivative is
devoid of a free thiol group.

44. The peptide composition of claim 43, wherein said Cys derivative is
selected from the group consisting of a protected Cys and Ser.

45. The peptide composition of claim 43, wherein said Cys derivative is
Ser.

46. The peptide composition of claim 45, wherein each said peptide
monomers independently has an amino acid sequence selected from the group
consisting of Asp-Cys-Ser-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID
NO:4; and Asp-Ser-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:5.

47. The peptide composition of claim 41, wherein at least one of said
peptide monomers comprises said amino acid sequence in which each of X1
and X2 is Cys.

48. The peptide composition of claim 47, wherein each of said peptide
monomers comprises the amino acid sequence
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:3.

49. The peptide composition of claim 41, wherein said two peptide
monomers are linked to one another by at least one disulfide bond.

50. The peptide composition of claim 41, wherein said two peptide
monomers are linked to one another by a single disulfide bond.

51. The peptide composition of claim 49, wherein said disulfide bond is
an intermolecular disulfide bond formed between two of said Cys residues.

52. The peptide composition of claim 41, wherein at least one of said two
peptide monomers consists of said amino acid sequence.

53. The peptide composition of claim 41, wherein a mol percentage of said
dimeric peptides in the composition is at least 90 mol percents.

54. The peptide composition of claim 41, wherein a mol percentage of said
dimeric peptides in the composition is at least 99 mol percents.

55. The peptide composition of claim 41, wherein a mol percentage of a
multimeric peptide, a monomeric peptide and/or a dimeric peptide other
than said dimeric peptide is lower than 1 mol percent.

56. A process of preparing the peptide composition of claim 41, the
process comprising: reacting two monomeric peptides, each independently
comprising the amino acid sequence: Asp-X1-X2-Asn-Tyr-Ile-Thr-X3
wherein: X1 is selected from the group consisting of Cys and a Cys
derivative; X2 is selected from the group consisting of Cys and a Cys
derivative; and X3 is selected from the group consisting of Arg and
Glu-Leu-Arg, provided that at least one of X1 and X2 is Cys, in the
presence of an oxidizing agent, thereby producing the peptide
composition.

57. The process of claim 56, wherein said reacting is performed under
conditions that favor intermolecular interactions between said monomeric
peptides.

58. The process of claim 56, wherein at least one of said monomeric
peptides comprises an amino acid sequence in which one of X1 and X2 is a
Cys derivative.

59. The process of claim 58, wherein said Cys derivative is Ser.

60. The process of claim 58, wherein said Cys derivative is a protected
Cys.

61. The process of claim 60, further comprising, prior to reacting said
two monomeric peptides with said oxidizing agent: reacting a monomeric
peptide which comprises an amino acid sequence in which X1 and X1 are
each Cys with a cysteine protecting group, to thereby obtain said
monomeric peptide is which each of X1 and X2 is said protected Cys.

62. The process of claim 61, further comprising, prior to reacting said
two monomeric peptides with said oxidizing agent: selectively removing
said cysteine protecting group, to thereby obtain a monomeric peptide is
which one of X1 and X2 is said protected Cys.

63. The process of claim 60, further comprising, prior to reacting said
two monomeric peptides with said oxidizing agent: reacting a monomeric
peptides which comprises an amino acid sequence in which X1 and X1 are
each Cys with a cysteine protecting group, under conditions that favor
selective protection, to thereby obtain said peptide monomer is which one
of X1 and X2 is said protected Cys.

64. The process of claim 56, wherein said oxidizing agent is iodine.

65. A pharmaceutical composition comprising the peptide composition of
claim 41, and a pharmaceutically acceptable carrier.

66. The pharmaceutical composition of claim 65, being packaged in a
packaging material an identified in print, in or on, said packaging
material, for use in the treatment of an allergic disorder.

68. The pharmaceutical composition of claim 65, wherein at least one of
said peptide monomers comprises said amino acid sequence in which one of
X1 and X2 is said Cys derivative.

69. The pharmaceutical composition of claim 65, wherein said Cys
derivative is devoid of a free thiol group.

70. The pharmaceutical composition of claim 69, wherein said Cys
derivative is selected from the group consisting of a protected Cys and
Ser.

71. The pharmaceutical composition of claim 70, wherein each said peptide
monomers independently has an amino acid sequence selected from the group
consisting of Asp-Cys-Ser-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID
NO:4; and Asp-Ser-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:5.

72. The pharmaceutical composition of claim 65, wherein at least one of
said peptide monomers comprises said amino acid sequence in which each of
X1 and X2 is Cys.

73. The pharmaceutical composition of claim 72, wherein each of said
peptide monomers comprises the amino acid sequence
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:3.

74. The pharmaceutical composition of claim 65, wherein said two peptide
monomers are linked to one another by at least one disulfide bond.

75. The pharmaceutical composition of claim 74, wherein said disulfide
bond is an intermolecular disulfide bond formed between two of said Cys
residues.

76. The pharmaceutical composition of claim 65, wherein at least one of
said two peptide monomers consists of said amino acid sequence.

77. The pharmaceutical composition of claim 65, wherein a mol percentage
of a multimeric peptide, a monomeric peptide and/or a dimeric peptide
other than said dimeric peptide is lower than 1 mol percent.

78. A method of treating an allergic disorder comprising administering to
a subject in need thereof a therapeutically effective amount of the
peptide composition of claim 41, thereby treating the allergic disorder.

Description:

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention, in some embodiments thereof, relates to
dimeric peptides based on the C-terminal sequence of human complement
C3a. The dimeric peptides inhibit secretory responses of mast cells and
basophils and accordingly may be useful in the treatment of allergic
disorders, such as asthma.

[0002] Mast cells and basophils play a central role in inflammatory and
immediate hypersensitivity reactions. Clustering of the type 1
Fcε receptors (FcεRI) present in the plasma membranes of
mast cells and basophils initiates a coupling network culminating in the
secretion of inflammatory mediators including histamine, serotonin,
proteases, leukotriens and several cytokines. The molecular mechanism of
signal transduction initiated by FcεRI clustering has been
intensively studied over the past few years. Lyn, a src family protein
tyrosine kinase (PTK) interacts with the β subunit of the receptor
complex and undergoes phosphorylation and activation as a result of
FcεRI clustering. Recruitment of Lyn to the immunoreceptor
tyrosine-based activation motif (ITAM)-phosphorylated receptor subunits
inter-alia results in activation of Syk PTK which in turn causes
phospholipase C-γ (PLC-γ) activation, hydrolysis of
phosphatidyl-inositide-4,5-bisphosphate (PIP2) and a transient rise in
free cytosolic [Ca2+]i. This in turn induces activation of protein kinase
C culminating eventually in the cells' secretory response.

[0003] Mast cell progenitors represent a single lineage, giving rise, upon
migration into different tissues to two distinct phenotypes; the
so-called serosal (connective tissue type) mastocytes residing in serosal
cavities, in the skin and respiratory tract; and the mucosal type mast
cells found mainly in regions exposed to potential pathogens such as the
gastrointestinal mucosal surfaces. Nevertheless, mast cell
tissue-dependent differentiation is reversible; fibroblast derived
factors change mucosal type mast cells into serosal ones, while IL-3
favors the mucosal phenotype. Besides tissue distribution, life span and
mediator content of their intracellular granules are also different. Both
types express FcεRI on their cell membrane, clustering of which
provokes the secretory response.

[0004] In contrast to the FEεRI-mediated triggering of mastocytes,
only serosal mast cells respond to the `peptidergic` stimuli. The latter
cells are experimentally modeled by rat peritoneal or human skin mast
cells. The peptidergic stimulus is triggered by exposure to polyamines or
cationic peptides such as substance P, or the complement activation
products C3a and C5a (Mousli et al., Immunopharmcol. 27: 1-11, 1995).
These complement-derived anaphylatoxins are among the most potent
peptidergic activators of (serosal) mast cells' secretory response. In
contrast, mucosal mast cells, such as the rat basophilic leukemia cell
line (RBL-2H3) do not respond to such cationic peptides. It was
demonstrated that C3a and some of its derivatives inhibit the
IgE-mediated degranulation of RBL-2H3 cells, while C5a has no effect on
this process (Erdei et al. Int. Immunol. 7: 1433-1439, 1995; Erdei et al.
Immunol. Lett. 68: 79-82, 1999).

[0005] However, the native intact C3a is not suitable as a potential
anti-allergic drug primarily because it contains an activating motif that
makes it anaphylatoxic to serosal mast cells, i.e., it is capable of
inducing mediator secretion from mast cells.

[0008] There remains an unmet need for compounds having improved efficacy
in preventing or treating allergic disorders.

[0009] Embodiments of the present invention relate to multimeric peptides,
particularly dimeric peptides, comprising peptide monomers derived from
and corresponding partially to the amino acid sequence at positions 55-64
of human complement component C3a useful for inhibit secretory responses
of mast cells and basophils and accordingly may be useful for the
treatment of allergic disorders, such as asthma. As discussed
hereinabove, the present inventors have previously designed and
successfully prepared and practiced monomeric peptides derived from and
corresponding partially to the amino acid sequence at positions 55-64 of
human complement component C3a (see, WO 2007/013083). These monomeric
peptides were prepared and used while maintaining a reduced form thereof,
namely, while not being subjected to oxidizing conditions where formation
of disulfide bridges are formed so as to produce dimeric and other
multimeric peptides.

[0010] The present inventors have tested the effect of combining peptide
monomers derived from and corresponding partially to the amino acid
sequence at positions 55-64 of human complement component C3a into
multimeric peptides, and have uncovered that dimeric peptides formed from
such monomeric peptides exhibit improved water solubility and stability
as compared to their building monomeric peptides, and hence exhibit
improved bioavailability. Moreover, the present inventors have uncovered
that dimeric peptides which are prepared such that a single dimeric
species is obtained exhibit an improved biological activity as compared
to both their building peptide monomers and a mixture of dimeric
peptides.

[0011] As demonstrated in the Examples section that follows, it has been
uncovered that the dimeric peptides disclosed herein are highly effective
in inhibiting FcεRI-mediated activation of mast cells and
basophils. It has further been uncovered that the dimeric peptides
described herein are capable of reducing allergic symptoms such as
passive systemic anaphylaxis in animal models.

[0012] According to an aspect of embodiments of the present invention
there is provided a peptide composition comprising a dimeric peptide, the
dimeric peptide comprising two peptide monomers, each independently
comprising the amino acid sequence:

Asp-X1-X2-Asn-Tyr-Ile-Thr-X3

[0013] wherein: [0014] X1 is selected from the group consisting of Cys
and a Cys derivative; [0015] X2 is selected from the group consisting of
Cys and a Cys derivative; and [0016] X3 is selected from the group
consisting of Arg and Glu-Leu-Arg, [0017] provided that at least one of
X1 and X2 is Cys,

[0018] wherein a mol percentage of the dimeric peptide in the composition
is at least 50 mol percents.

[0019] According to some embodiments of the invention, at least one of the
peptide monomers comprises the amino acid sequence in which one of X1 and
X2 is the Cys derivative.

[0020] According to some embodiments of the invention, each of the peptide
monomers comprises the amino acid in which one of X1 and X2 is the Cys
derivative.

[0021] According to some embodiments of the invention, the Cys derivative
is devoid of a free thiol group.

[0022] According to some embodiments of the invention, the Cys derivative
is selected from the group consisting of a protected Cys and Ser.

[0023] According to some embodiments of the invention, the Cys derivative
is Ser. Optionally, the Cys derivative is any other amino acid that does
not affect properties and activity of the peptide. An exemplary amino
acid is Thr.

[0024] According to some embodiments of the invention, each the peptide
monomers independently has an amino acid sequence selected from the group
consisting of Asp-Cys-Ser-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID
NO:4; and Asp-Ser-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:5.

[0025] According to some embodiments of the invention, at least one of the
peptide monomers comprises the amino acid sequence in which each of X1
and X2 is Cys.

[0026] According to some embodiments of the invention, each of the peptide
monomers comprises the amino acid in which each of X1 and X2 is Cys.

[0027] According to some embodiments of the invention, each of the peptide
monomers comprises the amino acid sequence
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:3.

[0028] According to some embodiments of the invention, the two peptide
monomers are linked to one another by at least one disulfide bond.

[0029] According to some embodiments of the invention, the two peptide
monomers are linked to one another by a single disulfide bond.

[0030] According to some embodiments of the invention, the disulfide bond
is an intermolecular disulfide bond formed between two of the Cys
residues.

[0031] According to some embodiments of the invention, at least one of the
two peptide monomers consists of the amino acid sequence.

[0032] According to some embodiments of the invention, each of the two
peptide monomers consists of the amino acid sequence.

[0033] According to some embodiments of the invention, a mol percentage of
the dimeric peptides in the composition is at least 90 mol percents.

[0034] According to some embodiments of the invention, a mol percentage of
the dimeric peptides in the composition is at least 99 mol percents.

[0035] According to some embodiments of the invention, a mol percentage of
a multimeric peptide, a monomeric peptide and/or a dimeric peptide other
than the dimeric peptide is lower than 1 mol percent.

[0036] According to some embodiments of the invention, the dimeric peptide
is characterized by an ordered beta sheet structure.

[0037] According to some embodiments of the invention, the dimeric peptide
comprises and anti-allergic activity.

[0038] According to another aspect of embodiments of the present invention
there is provided a process of preparing the peptide composition
described herein, the process comprising:

[0040] wherein: [0041] X1 is selected from the group consisting of Cys
and a Cys derivative; [0042] X2 is selected from the group consisting of
Cys and a Cys derivative; and [0043] X3 is selected from the group
consisting of Arg and Glu-Leu-Arg, provided that at least one of X1 and
X2 is Cys,

[0044] in the presence of an oxidizing agent, thereby producing the
peptide composition.

[0045] According to some embodiments of the invention, the reacting is
performed under conditions that favor intermolecular interactions between
the monomeric peptides.

[0046] According to some embodiments of the invention, the reacting is
performed in the presence of a solvent.

[0047] According to some embodiments of the invention, at least one of the
monomeric peptides comprises an amino acid sequence in which one of X1
and X2 is a Cys derivative.

[0048] According to some embodiments of the invention, the Cys derivative
is Ser.

[0049] According to some embodiments of the invention, the Cys derivative
is a protected Cys.

[0050] According to some embodiments of the invention, the process is
further comprising, prior to reacting the two monomeric peptides with the
oxidizing agent:

[0051] reacting a monomeric peptide which comprises an amino acid sequence
in which X1 and X1 are each Cys with a cysteine protecting group, to
thereby obtain the monomeric peptide is which each of X1 and X2 is the
protected Cys.

[0052] According to some embodiments of the invention, the process is
further comprising, prior to reacting the two monomeric peptides with the
oxidizing agent:

[0053] selectively removing the cysteine protecting group, to thereby
obtain a monomeric peptide is which one of X1 and X2 is the protected
Cys.

[0054] According to some embodiments of the invention, the process is
further comprising, prior to reacting the two monomeric peptides with the
oxidizing agent:

[0055] reacting monomeric peptides which comprises an amino acid sequence
in which X1 and X1 are each Cys with a cysteine protecting group, under
conditions that favor selective protection, to thereby obtain the peptide
monomer is which one of X1 and X2 is the protected Cys.

[0056] According to some embodiments of the invention, the oxidizing agent
is iodine.

[0057] According to an aspect of embodiments of the present invention
there is provided a peptide composition as described herein which is
being characterized for use in the treatment of an allergic disorder.

[0058] According to an aspect of embodiments of the present invention
there is provided a pharmaceutical composition comprising the peptide
composition as described herein, and a pharmaceutically acceptable
carrier.

[0059] According to some embodiments of the invention, the pharmaceutical
composition is being packaged in a packaging material an identified in
print, in or on, the packaging material, for use in the treatment of an
allergic disorder.

[0060] According to an aspect of embodiments of the present invention
there is provided a method of treating an allergic disorder comprising
administering to a subject in need thereof a therapeutically effective
amount of the peptide composition as described herein, thereby treating
the allergic disorder.

[0061] According to an aspect of embodiments of the present invention
there is provided a use of the peptide composition as described herein in
the manufacture of a medicament for treating an allergic disorder.

[0062] According to some embodiments of the invention, the allergic
disorder results from an IgE- or IgG-mediated (Type I or Type III)
hypersensitivity and/or FcεRI- or FcεR-induced secretory
response.

[0063] According to some embodiments of the invention, the allergic
disorder is mediated by a cell type selected from the group consisting of
mucosal-type mast cells, serosal-type mast cells and basophils.

[0065] According to some embodiments of the invention, the pulmonary
disease is asthma.

[0066] According to some embodiments of the invention, the allergic
dermatosis is urticaria.

[0067] Unless otherwise defined, all technical and/or scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
methods and materials similar or equivalent to those described herein can
be used in the practice or testing of embodiments of the invention,
exemplary methods and/or materials are described below. In case of
conflict, the patent specification, including definitions, will control.
In addition, the materials, methods, and examples are illustrative only
and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Some embodiments of the invention are herein described, by way of
example only, with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of illustrative
discussion of embodiments of the invention. In this regard, the
description taken with the drawings makes apparent to those skilled in
the art how embodiments of the invention may be practiced.

[0071] FIGS. 2A-F are graphs illustrating the inhibitory effect of the
dimeric peptides (FIGS. 2D-F) compared to their peptide monomers (FIGS.
2B-C) and full length peptide (FIG. 2A) on mast cell secretion. Data are
expressed as fraction of enzyme (β-hexosaminidase) secreted upon a
suboptimal stimulus by antigen (DNP11-BSA) from RBL-2H3 cells. The
average values of the inhibitory activity results of 6 independent
experiments (each performed in triplicates) are presented.

[0072]FIG. 3 is a bar graph illustrating inhibition by peptides of
degranulation of human basophils as monitored by flow-cytometric
measurement of the cell-surface CD63 expression upon
FcεRI-clustering. Data are expressed as percentage of
activation-induced CD63 expression obtained after a suboptimal stimulus.
Results shown are average values of 6 independent experiments. The final
concentration of the employed peptides was 200 μM.

[0073]FIG. 4 is a bar graph illustrating the inhibitory effect of
peptides of histamine-release induced by an in vivo challenge in mice.
Data shown are results of 3 experiments.

[0075]FIG. 6 illustrates the inhibition of CD63 expression by monomeric
or dimeric peptides with sequences derived from human complement
component C3a after FcεRI-clustering on human basophils. The
numbers above the small columns indicate the number of donors tested.

[0077] The present invention, in some embodiments thereof, relates to
dimeric peptides based on the C-terminal sequence of human complement
C3a. The dimeric peptides inhibit secretory responses of mast cells and
basophils and as such may be used to treat allergic disorders such as
asthma.

[0078] Before explaining at least one embodiment of the invention in
detail, it is to be understood that the invention is not necessarily
limited in its application to the details set forth in the following
description or exemplified by the Examples. The invention is capable of
other embodiments or of being practiced or carried out in various ways.

[0079] In the search for peptides capable of inhibiting mast cell
activity, the present inventors synthesized and characterized novel
peptides based on the complement activation product C3a.

[0080] In an attempt to generate peptides with well-defined structures,
while considering the presence of two cysteine residues in previously
reported peptide monomers derived from and corresponding partially to
human complement component C3a, (e.g., C3a9), the present inventors
synthesized C3a-based peptide derivatives that comprise a single cysteine
residue. Bound by this restriction, only a single dimer species can be
produced upon thiol oxidation, thereby eliminating the potential problem
of peptides that could be transformed into ill-defined structures by
e.g., non-selective dimer formation, intramolecular reactions and/or
oligomer formation due to the two cysteine thiol side chains present in
earlier peptides (e.g. C3a9).

[0081] The dimeric peptides disclosed in the present invention are derived
from the known human complement component C3a, a 77-mer polypeptide as
set forth in SEQ ID NO:1 of the sequence:

[0082] Particularly, the dimeric peptides disclosed herein are derived
from and corresponding partially to the amino acid sequence of positions
55-64 of human complement peptide C3a of the sequence:
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg (also referred to as C3a7) as set
forth in SEQ ID NO:2, to analogs, chemical derivatives, and salts thereof
capable of inhibiting mast cell and/or basophil activation. More
particularly, the dimeric peptides disclosed herein are derived from and
corresponding partially to the amino acid sequence of positions 55-64 of
human complement peptide C3a of the sequence:
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg (also referred to as C3a9) as set forth
in SEQ ID NO:3, to analogs, chemical derivatives, and salts thereof
capable of inhibiting mast cell and/or basophil activation.

[0083] While reducing the present invention to practice, the present
inventors prepared dimeric peptides of C3a9, have further synthesized two
peptides with modified C3a sequences, namely DSCNYITR (SEQ ID NO: 5) and
DCSNYITR (SEQ ID NO: 4) each having a single cysteine residue, and
generated well-defined dimers therefrom.

[0084] As illustrated in the Examples section below, the present inventors
showed that both these dimers are soluble in water and exhibit circular
dichroism (Table 3 and FIGS. 1A-D). In addition, the present inventors
showed that both dimers prevent degranulation of mast cells and human
basophils, as measured by monitoring activity of released
β-hexoseaminidase from a rat mucosal-type mast cell line (FIGS.
2A-F) and analysis of expression of CD63, a granular membrane protein on
the surface of human basophils by flow cytometry (FIG. 3). Finally, the
present inventors demonstrated that both dimers prevent in vivo histamine
release in mice following passive systemic anaphylaxis (FIG. 4).

[0085] These findings demonstrate an improved pharmacological profile of
dimeric peptides derived from and corresponding partially to the amino
acid sequence of positions 55-64 of human complement peptide C3a, and
accordingly, demonstrate an improved effect of these dimeric peptides in
treating allergic disorders.

[0086] Thus, according to one aspect of the present invention there is
provided a peptide composition comprising a dimeric peptide, said dimeric
peptide comprising two peptide monomers, each independently comprising
the amino acid sequence:

Asp-X1-X2-Asn-Tyr-Ile-Thr-X3

[0087] wherein: [0088] X1 is selected from the group consisting of Cys
and a Cys derivative; [0089] X2 is selected from the group consisting of
Cys and a Cys derivative; and [0090] X3 is selected from the group
consisting of Arg and Glu-Leu-Arg, provided that at least one of X1 and
X2 is Cys,

[0091] wherein a mol percentage of said dimeric peptide in the composition
is at least 50 mol percents.

[0092] As used herein, the phrase "peptide composition" describes a
product of a peptide synthesis. As is well known in the art, a product of
a peptide synthesis typically comprises peptide(s) having a desired
sequence, as defined herein, contaminated by peptides having an undesired
sequence, as defined herein.

[0093] In the context of embodiments of the present invention, the phrase
"peptide composition" describes a product of a peptide synthesis in which
peptide monomers are subjected to reaction conditions that allow or
promote bond formation between two amino acids.

[0094] The phrase "dimeric peptide" refers to a peptide having two peptide
monomers associated covalently by covalent interactions. Thus, dimeric
peptides, as used herein, include at least one intermolecular covalent
bond. Dimeric peptides, however, can further exhibit non-covalent bonds,
both intramolecular and intermolecular non-covalent bonds.

[0095] In exemplary embodiments, a peptide composition is a product or a
peptide synthesis in which monomeric peptides are subjected to reaction
conditions that allow or promote formation of one or more disulfide
bridge between two cysteine residues.

[0096] The phrase "peptide composition" encompasses both, a product of a
peptide synthesis isolated from the reaction medium in which the peptide
was prepared, and such a product contained within the reaction medium in
which the peptide was prepared. This phrase further encompasses a product
of a peptide synthesis which is isolated from the reaction medium in
which the peptide was prepared and is further purified, such that a
peptide having the desired amino acid sequence is separated from peptides
having an undesired sequence.

[0097] In the context of the present embodiments, the phrase "a peptide
having a desired sequence" describes a dimeric peptide that comprises two
peptide monomers which are bound to one another via pre-determined
intermolecular bonds (e.g., one or more pre-determined disulfide bridges)
between pre-determined amino acids in each peptide monomer.

[0098] The phrase "a peptide having an undesired sequence" describes any
of the following: (i) peptide monomers which have a secondary structure
other than the monomeric peptides used for synthesizing the peptide
product; (ii) dimeric peptides that comprises two peptide monomers which
are bound to one another via bonds between one or more amino acids in one
peptide monomer and one or more amino acids in the other peptide monomer,
and which have a different primary structure as compared with a dimeric
peptide having a desired sequence, as defined herein; and (iii)
oligomeric peptides, comprising 3 or more peptides that are bound to one
another.

[0099] Thus, the phrase "a peptide having an undesired sequence" in the
context of embodiments of the invention, therefore encompasses monomeric
and/or oligomeric peptides, and dimeric peptides that differ from dimeric
peptides having a desired sequence by the position, chemical structure
and/or number of amino acid residues that participate in the
intermolecular bond(s) that links two peptide monomers in the dimeric
peptide.

[0100] A peptide composition, as described herein, is typically in a form
of a solid or a solution. Solid compositions can be, for example,
powdered or lyophilized. The composition can further be bound to a solid
support.

[0101] Herein throughout, the phrase "peptide monomer" is used to describe
a peptide monomer that forms a part of a dimeric peptide, unless
otherwise indicated. The phrase "monomeric peptide" is used to describe a
peptide in a monomeric form thereof (which does not form a part of a
dimeric or oligomeric peptide), unless otherwise indicated.

[0102] According to embodiments of the invention, the peptide composition
comprises a dimeric peptide formed from two peptide monomers, each
peptide monomer being derived from and corresponding partially to the
sequence of amino acids 55-64 of human complement C3a, and each
comprising at least one cysteine residue, such that the dimeric peptide
comprises one or more intermolecular disulfide bridges formed between two
cysteine residues.

[0103] Thus, in some embodiments of the invention, each of the peptide
monomers comprises an amino acid sequence as described herein, in which
at least one of X1 and X2 is a cysteine residue, such that the peptide
monomers are covalently linked to one another via a disulfide bridge
formed between a cysteine residue in one peptide monomer and a cysteine
residue in another peptide monomer.

[0104] In some embodiments, the peptide monomers forming the dimeric
peptide are covalently linked to one another via a single disulfide
bridge, as further detailed hereinbelow.

[0105] In some embodiments, the peptide monomers forming the dimeric
peptide are covalently linked to one another via two disulfide bridges,
as further detailed hereinbelow.

[0106] Hereinthroughout, the phrases "disulfide bridge" and "disulfide
bond" are used interchangeably, and describe a --S--S-- bond.

[0107] It is to be noted that the disulfide bridges referred to herein
relate to the part of the dimeric peptide that is formed by the portions
of the peptide monomers that have the amino acid sequence described
herein. Thus, in cases where the one or both peptide monomers comprises
additional amino acids, the dimeric peptides may have additional
intermolecular bonds, including additional disulfide bridges, linking two
amino acids other than those forming the amino acid sequence described
herein.

[0108] As further detailed hereinbelow, the peptide composition described
herein was designed and successfully prepared via a directed synthesis,
for obtaining a desired dimeric peptide in which one or two
intermolecular disulfide bridge is formed between the two peptide
monomers, at a predetermined position (namely, between two predetermined
cysteines).

[0109] The peptide compositions described herein is therefore such that a
majority of the molecules in the composition are a dimeric peptide of a
desired, pre-determined chemical structure.

[0110] Thus, in some embodiments, the peptide composition described herein
comprises at least 50 mol percents of the dimeric peptide, at least 60
mol percents of the dimeric peptide, at least 70 mol percents of the
dimeric peptide, at least 80 mol percents of the dimeric peptide, at
least 90 mol percents of the dimeric peptide, at least 95 mol percents of
the dimeric peptide, at least 98 mol percents of the dimeric peptide, at
least 99 mol percents of the dimeric peptide, at least 99.1 mol percents,
at least 99.2 mol percents, at least 99.3 mol percents, at least 99.4 mol
percents, at least 99.5 mol percents, at least 99.6 mol percents, at
least 99.7 mol percents, at least 99.8 mol percents, at least 99.9 mol
percents, at least 99.95 mol percents, at least 99.96 mol percents, at
least 99.97 mol percents, at least 99.98 mol percents, at least 99.99 mol
percents, and even consists of the dimeric peptide, such that it includes
100 mol percents of the dimeric peptide (e.g., an isolated dimeric
peptide).

[0111] By "mol percents" it is meant the partial number of molecules of
the dimeric peptide out of the total number of molecules in the peptide
composition.

[0112] As is well recognized by those skilled in the art, the synthesis of
peptides comprising disulphide bridges is challenging since it is
difficult to ensure that the correct cysteine residues combine to form
the desired disulphide bridges.

[0113] Thus, for example, in peptide compositions as described herein, in
which one or both of the peptide monomers comprise two cysteine residues
(as X1 and X2), dimeric peptides can be formed with each of the cysteine
residues, as exemplified hereinafter, so as to form a mixture of dimeric
species. In addition to the mixture of such dimeric species, oligomeric
peptides can be formed. Furthermore, peptide monomers in which the two
cysteines are combined intramolecularly can be formed.

[0114] However, in some embodiments, the peptide compositions described
herein can be such that include dimeric peptides in which selected
cysteine residues combine to form a disulfide bridge.

[0115] In some embodiments, the peptide compositions described herein thus
consist of a single (homo or hetero) dimeric species, as defined herein,
in which a selected cysteine residue in each peptide monomer (X1 or X2,
as described herein) participates in the formed disulfide bridge(s).

[0116] In some embodiments of the invention, a mol percentage of a
oligomeric peptide, a monomeric peptide and/or a dimeric peptide other
than the dimeric peptide described herein is lower than 1 mol percent,
and can be lower than 0.5 mol percent, lower than 0.1 mol percent, lower
than 0.05 mol percent, lower than 0.01 mol percent, and even lower.

[0117] In some embodiments, the peptide composition is devoid of
oligomeric peptides, monomeric peptides and dimeric peptides other than
the dimeric peptides described herein.

[0118] The phrase "oligomeric peptide" as used herein, describes a peptide
formed from three or more peptide monomers that are covalently linked
therebetween, optionally via disulfide bridges. The phrase "dimeric
peptide other than said dimeric peptide" describes a dimeric peptide
which comprises intermolecular bonds other than those described herein.
Examples of such other dimeric peptide include, but are not limited to,
dimeric peptides that differ from one another by the number and/or
position of the disulfide bonds, a dimeric peptide which includes bonds
other than disulfide bridges, and any other dimeric peptide not
encompassed by embodiments of the invention.

[0119] In some embodiments, the peptide composition is such that the amino
acid sequence of each of the two peptide monomers in the dimeric peptide
are the same, forming a homodimeric peptide. In cases where one of or
both monomeric peptides that combine to form a homodimeric peptide
comprise two cysteine residues, several homodimeric species, which differ
from one another by the cysteine residues that participate in the
disulfide bridge, can be formed.

[0120] In some embodiments, the peptide composition is such that the amino
acid sequence of each of the two peptide monomers in the dimeric peptide
is different, forming a heterodimeric peptide. Heterodimeric peptides can
also include heterodimeric species, as detailed hereinafter.

[0121] In some embodiments, in one or both peptide monomers, each of X1
and X2 is a cysteine residue.

[0122] In some embodiments, a peptide monomer in which each of X1 and X2
is a cysteine residue comprises the amino acid sequence
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg, as set forth in SEQ ID NO:3, also
referred to herein as C3a9.

[0123] In some embodiments, a peptide monomer in which each of X1 and X2
is a cysteine residue comprises the amino acid sequence
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg, as set forth in SEQ ID NO:2,
also referred to as C3a7.

[0124] In embodiments where in one or both peptide monomers, each of X1
and X2 is a cysteine residue, the peptide composition can comprise
dimeric peptides in which intermolecular disulfide bridges are formed
between any combinations of the two cysteine residues, as is further
detailed hereinbelow.

[0125] Dimeric peptides that are formed from two peptide monomers in which
each of X1 and X2 is a cysteine residue, and which have the same amino
acid sequences, may form different homodimeric species.

[0126] By "homodimeric species" it is meant that a dimeric peptide
comprises two monomeric peptides that have identical amino acid
sequences, and one or two disulfide bridges, each linking t a cysteine
residue at one position of one monomer and a cysteine residue at one
position of the other monomer. That is, a homodimeric species may have a
disulfide bridge linking X1 in one monomer to X1 in another monomer; a
disulfide bridge linking X1 in one monomer to X2 in another monomer; a
disulfide bridge linking X2 in one monomer to X2 in another monomer; two
disulfide bridges linking X1 in one monomer to X1 in another monomer and
X2 in one monomer to X2 in another monomer; and two disulfide bridges
linking X1 in one monomer to X2 in another monomer and X2 in one monomer
to X1 in another monomer.

[0127] For example, dimeric peptides formed from two peptide monomers
having SEQ ID NO:3 (C3a9), can have the following structures:

[0129] In some embodiments, the dimeric peptide comprises a mixture of
homodimeric species.

[0130] In some embodiments, the dimeric peptide comprises a single
homodimeric species.

[0131] In some embodiments, in at least one of the peptide monomers, one
of X1 and X2 is a cysteine derivative.

[0132] As used herein, a "cysteine derivative" or "a Cys derivative"
describes a structural analog of cysteine, which does not comprise a free
thiol group.

[0133] According to some embodiments of the invention, cysteine
derivatives have the following structure:

##STR00002##

[0134] wherein Y is other than thiol.

[0135] As used herein, the term "thiol" describes as --SH group.

[0136] Thus, in some embodiments, the Cys derivative is devoid of a free
thiol group.

[0137] In some embodiments, Y is a --OH group, such that the cysteine
derivative is a Serine residue.

[0138] Exemplary peptide monomers in which the cysteine derivative is Ser
have, as non-limiting example, the amino acid sequence
Asp-Cys-Ser-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:4 (also
referred to herein as Monomer A or 5508); or the amino acid sequence
Asp-Ser-Cys-Asn-Tyr-Ile-Thr-Arg as set forth in SEQ ID NO:5 (also
referred to herein as Monomer B, or 5513).

[0139] In some embodiments, Y is a Cysteine protecting group, such that
the Cys derivative is a protected Cys residue.

[0140] The phrase "protected Cys residue" describes a cysteine residue in
which the free thiol group at the side chain is derivatized by a chemical
group that can be removed, so as to regenerate, under certain conditions,
a free thiol group. Such chemical groups are known as cysteine protecting
groups.

[0143] In some embodiments, the Cys derivative is an amino acid or an
analog thereof which do not affect properties such as water solubility,
hydrophilicity, and the desired activity, of the peptide monomer and the
dimer formed therefrom. An exemplary such amino acid is Thr. Exemplary
such peptide monomers have an amino acid sequence as set forth is SEQ ID
NO:6 and SEQ ID NO:7.

[0144] In embodiments where one peptide monomer has the amino acid
sequence as described herein in which both X1 and X2 are cysteine and the
other monomer has the amino acid sequence as described herein in which
one of X1 and X2 is cysteine and the other is a cysteine derivative, as
described herein, the dimeric peptide is a heterodimer. In such a
heterodimer, two heterodimer species can be formed, by the formation of a
disulfide bridge between the single cysteine residue in one monomer and
one of the two cysteine residues in the other monomer.

[0145] For example, in cases where one monomer has the amino acid sequence
as set forth in SEQ ID NO:3 (C3a9), and one monomer has the amino acid
sequence as set forth in SEQ ID NO:4 (Monomer A), the following
heterodimer species can be formed:

##STR00003##

[0146] In cases where one monomer has the amino acid sequence as set forth
in SEQ ID NO:3 (C3a9), and one monomer has the amino acid sequence as set
forth in SEQ ID NO:5 (Monomer B), the following heterodimer species can
be formed:

##STR00004##

[0147] In some embodiments, in each of the peptide monomers forming the
dimeric peptide, one of X1 and X2 is cysteine and the other is a cysteine
residue, as described herein.

[0148] Such dimeric peptides can be heterodimeric peptides, where the
peptide monomers differ from one another by the position of the cysteine
residue, and/or by the nature of the cysteine derivative. Such dimeric
peptides can alternatively be homodimeric.

[0149] An exemplary such heterodimeric peptide is:

##STR00005##

[0150] Exemplary homodimeric peptides include:

##STR00006##

[0151] In some embodiments, the dimeric peptides described herein consist
of the amino acid sequence set forth hereinabove, as exemplified
hereinabove.

[0152] Alternatively, the dimeric peptides may comprise the amino acid
sequence set forth hereinabove, and may further comprise additional amino
acid residues.

[0153] Accordingly, in each of the exemplary structures set forth
hereinabove for dimeric peptides, additional amino acid residues can be
included in one or both peptide monomers. Such additional amino acid
residues may render the exemplary homodimeric peptides and homodimeric
species, heterodimeric peptides and heterodimeric species, respectively,
if the additional amino acid residues are not identical in both peptide
monomers.

[0154] In some embodiments, the dimeric peptides described herein are
characterized by an ordered beta sheet structure, as is further detailed
hereinunder.

[0155] As demonstrated in the Examples section that follows, and in FIGS.
1A-D, the ordered beta sheet structure was determined by circular
dichroism measurements.

[0156] The phrase "circular dischroism", as used herein and known in the
art, describes the differential absorption left-handed and right-handed
circularly polarized light.

[0157] As further demonstrated in the Examples section that follows, and
is detailed hereinunder, the peptide compositions described herein
exhibit anti-allergic activity.

[0158] As would be appreciated by any person skilled in the art, the
anti-allergic activity exhibited by such dimeric peptides, and
particularly an improved anti-allergic activity as compared with the
previously described monomeric peptides, is non-trivial, since in the
formed dimeric peptides, at least one reactive amino acid (namely, an
amino acid that forms a part of an active site of the previously
described monomeric peptide) is chemically modified, and yet, the
biological activity is maintained and even improved.

[0159] As would further be appreciated, the chemical modification relates
to (i) the formation of a disulfide bridge (oxidation of a Cys residue);
and (ii) the replacement of Cys by a Cys derivative.

[0160] Further according to an aspect of embodiments of the present
invention there is provided a process of preparing the peptide
composition as described herein. The process is effected by reacting two
monomeric peptides, each independently comprising the amino acid
sequence:

Asp-X1-X2-Asn-Tyr-Ile-Thr-X3

[0161] wherein: [0162] X1 is selected from the group consisting of Cys
and a Cys derivative; [0163] X2 is selected from the group consisting of
Cys and a Cys derivative; and [0164] X3 is selected from the group
consisting of Arg and Glu-Leu-Arg, [0165] provided that at least one of
X1 and X2 is Cys, as described herein, in the presence of an oxidizing
agent.

[0166] The oxidizing agent is selected so as to promote formation of a
disulfide bridge between two cysteine residues.

[0167] Exemplary oxidizing agents include iodine, preferably in the form
of an aqueous solution of iodine, and any other oxidizing agents known in
the art as promoting formation of a disulfide bridge between 2 cysteine
residues.

[0168] In some embodiments, reacting the monomeric peptides is performed
under conditions that favor intermolecular interactions between the
peptide monomers.

[0169] Thus, the peptide compositions described herein are prepared under
conditions that do not favor, and even do not enable, an intramolecular
reaction, even in the presence of an oxidizing agent.

[0170] As known in the art, intramolecular reactions are often favored
when a reaction mixture is diluted to such an extent where intermolecular
interactions between the reacting molecules are not energetically
favored, whereby intramolecular interactions of one or more reactants (if
feasible) are energetically favored. Accordingly, in some embodiments,
reacting the monomeric peptides in the presence of an oxidizing agent is
performed in a solution, namely, in the presence of a solvent, whereby
the concentration of each of the monomeric peptides in the solution is
such that intramolecular interactions are not favored, and intermolecular
interactions are favored.

[0171] Thus, in some embodiments, a concentration of each of said
monomeric peptides in said solvent is at least 20 μM, at least 50
μM, at least 100 μM, at least 200 μM, at least 300 μM, at
least 400 μM, at least 500 μM, and can be even 1 mM.

[0172] It is to be noted, however, that a concentration of a monomeric
peptide in the solvent at which intermolecular interactions are favored
depends also on the type, 22 reactivity and concentration of the
oxidizing agent, on the chemical structure of the peptide, and/or on the
type of solvent.

[0173] As noted hereinabove, the synthesis of peptides comprising
disulphide bridges is challenging since it is difficult to ensure that
the correct cysteine residues combine to form the desired disulphide
bridges.

[0174] As further known in the art, in a concentrated reaction mixture,
intermolecular reactions are favored, yet, such conditions further favor
the formation of numerous species of dimeric peptides and even oligomeric
peptides.

[0175] In order to obtain defined dimeric peptides, in which the cysteine
residues that combine to form a disulfide bridge are pre-determined, the
present inventors utilized various methodologies.

[0176] In one methodology, monomeric peptides comprising an amino acid
sequence as set forth hereinabove, in which in one or both monomeric
peptide(s) at least one of X1 and X2 is a Cys derivative, as defined
herein, were used.

[0177] A Cys derivative, as described herein, is devoid of a free thiol
group and hence do not participate in the formation of a disulfide
bridge. Monomeric peptides which comprise a Cys derivative include only a
single Cys residue that participate in the formation of a disulfide
bridge, and the formation of numerous dimeric species is avoided or at
least reduced.

[0178] Thus, in some embodiments, at least one of the monomeric peptides
used for forming the dimeric peptide comprises a Ser residue as X1 or X2,
thus forming dimeric peptide with a disulfide bond only between the
remaining Cys residues.

[0179] In some embodiments, two monomeric peptides, each comprising a Ser
residue as X1 or X2 were used, thus forming dimeric peptide with a
disulfide bond only between the remaining two Cys residues.

[0180] In another methodology, monomeric peptides which comprise two Cys
residues are selectively protected, so as to obtain one Cys residue and
one protected Cys residue, as X1 and X2 in the amino acid sequence
described herein.

[0181] In another methodology, peptide monomers which comprise two
cysteine residues are protected so as obtain two protected Cys residues,
and are thereafter subjected to selective deprotection, so as to obtain
one Cys residue and one protected Cys residue, as X1 and X2.

[0182] The latter methodology can be further used to selectively form
dimeric peptides with two disulfide bridges. Thus, upon obtaining two
protected Cys residues, the monomeric peptides are subjected to
sequential deprotection and oxidation procedures, under such conditions
that a first protected Cys residue is deprotected in each monomeric
peptide, a first oxidation is thereafter effected so as to form a first
disulfide bridge between pre-selected Cys residues, and then a second Cys
residue is deprotected, under conditions that do not allow reduction of
the first disulfide bridge, and a second oxidation is thereafter
effected, so as to form a second disulfide bridge.

[0183] Using any of these methodologies thus enables to obtain dimeric
peptides in which one or two disulfide bridge(s) is formed between
pre-selected cysteine residues.

[0184] It is to be noted that a process as described herein can be
utilized also for obtaining pre-selected homodimeric or heterodimeric
species. For example, a solution of a monomeric peptide which comprises
two cysteine residues can be treated, as described hereinabove, such that
one of the cysteine residues is selectively protected, and is then
subjected to oxidation, as described herein. Alternatively, two solutions
of a monomeric peptide which comprises two cysteine residues are
prepared, and each solution is treated differently, so as to obtain a
different cysteine residue that is selectively protected, and in then
subjected to oxidation. Further alternatively, a solution of a monomeric
peptide which comprises two cysteine residues can be treated, as
described hereinabove, such that one of the cysteine residues is
selectively protected, and this solution is then reacted with a peptide
monomer is which one of X1 and X2 is Ser.

[0185] It is to be noted that in cases where a protected Cys residue is
utilized, deprotection can be performed once the dimeric peptide is
obtained, so as to regenerate a Cys residue.

[0186] The monomeric peptides utilized for forming the dimeric peptides
described herein can be synthesized using methods well known in the art,
including chemical synthesis and recombinant DNA technology. Synthesis
may be performed by solid phase peptide synthesis described by Merrifield
(see J. Am. Chem. Soc., 85:2149, 1964). Alternatively, the peptide
monomers can be synthesized using standard solution methods (see, for
example, Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag,
1984). Preferably, the peptides of the invention are synthesized by solid
phase peptide synthesis as exemplified herein below (Example 1).

[0187] Cysteine protecting groups and methods of utilizing same for
forming protected Cysteines are also known in the art. Any known
methodology for forming a protected Cys residue and for deprotecting a
protected Cys residue, either selectively or non-selectively, is
contemplated herein. Particularly useful are methods for deprotecting a
protected Cys residue under conditions that do not enable reduction of a
disulfide bridge.

[0188] Exemplary methodologies are described in U.S. Pat. No. 6,906,171,
which is incorporated by reference as if fully set forth herein.

[0189] The term "alkyl", as used herein, describes a saturated aliphatic
hydrocarbon including straight chain and branched chain groups.
Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a
numerical range; e.g., "1-20", is stated herein, it implies that the
group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon
atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More
preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms.
Most preferably, unless otherwise indicated, the alkyl is a lower alkyl
having 1 to 4 carbon atoms. The alkyl group may be substituted or
unsubstituted.

[0190] The term "cycloalkyl" describes an all-carbon monocyclic or fused
ring (i.e., rings which share an adjacent pair of carbon atoms) group
where one or more of the rings does not have a completely conjugated
pi-electron system. The cycloalkyl group may be substituted or
unsubstituted.

[0191] The term "heteroalicyclic" describes a monocyclic or fused ring
group having in the ring(s) one or more atoms such as nitrogen, oxygen
and sulfur. The rings may also have one or more double bonds. However,
the rings do not have a completely conjugated pi-electron system. The
heteroalicyclic may be substituted or unsubstituted. Representative
examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane,
morpholino and the like.

[0192] The term "aryl" describes an all-carbon monocyclic or fused-ring
polycyclic (i.e., rings which share adjacent pairs of carbon atoms)
groups having a completely conjugated pi-electron system. The aryl group
may be substituted or unsubstituted.

[0193] The term "heteroaryl" describes a monocyclic or fused ring (i.e.,
rings which share an adjacent pair of atoms) group having in the ring(s)
one or more atoms, such as, for example, nitrogen, oxygen and sulfur and,
in addition, having a completely conjugated pi-electron system. Examples,
without limitation, of heteroaryl groups include pyrrole, furane,
thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,
quinoline, isoquinoline and purine. The heteroaryl group may be
substituted or unsubstituted.

[0195] The term "haloalkyl" describes an alkyl group as defined above,
further substituted by one or more halide.

[0196] The term "sulfate" describes a --O--S(═O)2--OR' group,
where R' can be hydrogen, alkyl, cycloalkyl or aryl, as defined herein.

[0197] The term "thiosulfate" describes a --O--S(═S)(═O)--OR'
group, where R' is as defined hereinabove.

[0198] The term "sulfoxide" or "sulfinyl" describes a --S(═O)R' group,
where R' is as defined hereinabove.

[0199] The term "sulfonate" describes a --S(═O)2--R' group, where R'
is as defined herein.

[0200] The term "sulfonamide" describes a --S(═O)2--NR'R'' group
or a R'S(═O)2--NR''-- group, where R' is as defined herein and
R'' is as defined herein for R'.

[0201] The term "phosphonate" describes a --P(═O)(OR')(OR'') group,
with R' and R'' as defined herein.

[0202] The term "thiophosphonate" describes a --P(═S)(OR')(OR'')
group, with R' and R'' as defined herein.

[0203] The term "phosphinyl" describes a --PR'R'' end group or a --PR'--
linking group, as these phrases are defined hereinabove, with R' and R''
as defined hereinabove.

[0204] The term "carbonyl" or "carbonate" as used herein, describes a
--C(═O)--R' group, with R' as defined herein.

[0205] The term "thiocarbonyl " as used herein, describes a
--C(═S)--R' group, with R' as defined herein.

[0206] The term "hydroxyl" describes a --OH group.

[0207] The term "alkoxy" describes both an --O-alkyl and an --O-cycloalkyl
group, as defined herein.

[0208] The term "aryloxy" describes both an --O-aryl and an --O-heteroaryl
group, as defined herein.

[0209] The term "thioalkoxy" describes both a --S-alkyl group, and a
--S-cycloalkyl group, as defined herein.

[0210] The term "thioaryloxy" describes both a --S-aryl and a
--S-heteroaryl group, as defined herein.

[0211] The term "cyano" describes a --C≡N group.

[0212] The term "isocyanate" describes an --N═C═O group.

[0213] The term "nitro" describes an --NO2 group.

[0214] The term "acyl halide" describes a --(C═O)R'''' group wherein
R'''' is halide, as defined hereinabove.

[0215] The term "carboxylate" describes a --C(═O)--OR' group or a
--OC(═O)R' group, where R' is as defined herein.

[0216] The term "thiocarboxylate" describes a --C(═S)--OR' group or a
--OC(═S)R' group, where R' is as defined herein.

[0217] The term "carbamate" describes an R''OC(═O)--NR'-- group or an
--OC(═O)--NR'R'' group, with R' and R'' as defined herein.

[0218] The term "thiocarbamate" describes a --OC(═S)--NR'R'' group or
a R''OC(═S)NR'-- group, with R' and R'' as defined herein.

[0219] The term "urea", which is also referred to herein as "ureido",
describes a --NR'C(═O)--NR''R''' group or a --NR'C(═O)--NR''--
linking group, where R' and R'' are as defined herein and R''' is as
defined herein for R' and R''.

[0220] The term "thiourea", which is also referred to herein as
"thioureido", describes a --NR'--C(═S)--NR''R''' group, with R', R''
and R''' as defined herein.

[0221] The term "amide" describes a R'C(═O)--NR''-- group or a
R'R''NC(═N)-- group, where R' and R'' are as defined herein.

[0222] The term "guanidine" describes a --R'NC(═N)--NR''R''' group,
where R', R'' and R''' are as defined herein.

[0223] The present invention further encompasses pharmaceutically
acceptable salts, prodrugs, solvates and hydrates of any of the
monomeric, dimeric and multimeric peptides described herein.

[0224] The phrase "pharmaceutically acceptable salt" refers to a charged
species of the parent compound and its counter ion, which is typically
used to modify the solubility characteristics of the parent compound
and/or to reduce any significant irritation to an organism by the parent
compound, while not abrogating the biological activity and properties of
the administered compound.

[0227] As used herein, the term "prodrug" refers to an agent, which is
converted into the active compound (the active parent drug) in vivo.
Prodrugs are typically useful for facilitating the administration of the
parent drug. They may, for instance, be bioavailable by oral
administration whereas the parent drug is not. The prodrug may also have
improved solubility as compared with the parent drug in pharmaceutical
compositions. Prodrugs are also often used to achieve a sustained release
of the active compound in vivo.

[0228] The term "solvate" refers to a complex of variable stoichiometry
(e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a
solute (the peptide) and a solvent, whereby the solvent does not
interfere with the biological activity of the solute. Suitable solvents
include, for example, ethanol, acetic acid and the like.

[0229] The term "hydrate" refers to a solvate, as defined hereinabove,
where the solvent is water.

[0230] The term "peptide" as used herein refers to a polymer of natural or
synthetic amino acids, encompassing native peptides (either degradation
products, synthetically synthesized polypeptides or recombinant
polypeptides) and peptidomimetics (typically, synthetically synthesized
peptides), as well as peptoids and semipeptoids which are peptide
analogs, which may have, for example, modifications rendering the
peptides even more stable while in a body or more capable of penetrating
into cells.

[0231] Such modifications include, but are not limited to N terminus
modification, C terminus modification, polypeptide bond modification,
including, but not limited to, CH2--NH, CH2--S,
CH2--S═O, O═C--NH, CH2--O, CH2--CH2,
S═C--NH, CH═CH or CF═CH, backbone modifications, and residue
modification. Methods for preparing peptidomimetic compounds are well
known in the art and are specified, for example, in Quantitative Drug
Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press
(1992), which is incorporated by reference as if fully set forth herein.
Further details in this respect are provided hereinunder.

[0235] In addition to the above, the polypeptides of the present invention
may also include one or more modified amino acids or one or more
non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc),
other than those described hereinabove.

[0236] As used herein in the specification and in the claims section below
the term "amino acid" or "amino acids" is understood to include the 20
naturally occurring amino acids;

[0237] those amino acids often modified post-translationally in vivo,
including, for example, hydroxyproline, phosphoserine and
phosphothreonine; and other unusual amino acids including, but not
limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid" includes
both D- and L-amino acids (stereoisomers).

[0238] Tables 1 and 2 below list naturally occurring amino acids (Table 1)
and non-conventional or modified amino acids (Table 2) which can be used
with the present invention.

[0239] The N and C termini of the peptides of the present invention may be
protected by function groups. Suitable functional groups are described in
Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and
Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated
herein by reference. Preferred protecting groups are those that
facilitate transport of the compound attached thereto into a cell, for
example, by reducing the hydrophilicity and increasing the lipophilicity
of the compounds.

[0240] These moieties can be cleaved in vivo, either by hydrolysis or
enzymatically, inside the cell. Hydroxyl protecting groups include
esters, carbonates and carbamate protecting groups. Amine protecting
groups include alkoxy and aryloxy carbonyl groups, as described above for
N-terminal protecting groups. Carboxylic acid protecting groups include
aliphatic, benzylic and aryl esters, as described herein for C-terminal
protecting groups. In one embodiment, the carboxylic acid group in the
side chain of one or more glutamic acid or aspartic acid residue in a
peptide of the present invention is protected, preferably with a methyl,
ethyl, benzyl or substituted benzyl ester.

[0242] The carboxyl group at the C-terminus of the compound can be
protected, for example, by an amide (i.e., the hydroxyl group at the
C-terminus is replaced with --NH2, --NHR2 and
--NR2R3) or ester (i.e. the hydroxyl group at the C-terminus is
replaced with --OR2). R2 and R3 are independently an
aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a
substituted aryl group. In addition, taken together with the nitrogen
atom, R2 and R3 can form a C4 to C8 heterocyclic ring with from
about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur.
Examples of suitable heterocyclic rings include piperidinyl,
pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of
C-terminal protecting groups include --NH2, --NHCH3, --N(CH3)2,
--NH(ethyl), --N(ethyl)2, --N(methyl)(ethyl), --NH(benzyl),
--N(C1-C4 alkyl)(benzyl), --NH(phenyl), --N(C1-C4 alkyl)(phenyl),
--OCH3, --O-(ethyl), --O-(n-propyl), --O-(n-butyl),
--O-(iso-propyl), --O-(sec-butyl), --O-(t-butyl), --O-benzyl and
--O-phenyl.

[0243] The peptides of the present embodiments may also comprise non-amino
acid moieties, such as for example, hydrophobic moieties (various linear,
branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon
derivatives) attached to the peptides; various protecting groups,
especially where the compound is linear, which are attached to the
compound's terminals to decrease degradation. Chemical (non-amino acid)
groups present in the compound may be included in order to improve
various physiological properties such; decreased degradation or
clearance; decreased repulsion by various cellular pumps, improve
immunogenic activities, improve various modes of administration (such as
attachment of various sequences which allow penetration through various
barriers, through the gut, etc.); increased specificity, increased
affinity, decreased toxicity and the like.

[0244] According to one embodiment, the peptides of the present
embodiments are attached to a sustained-release enhancing agent.
Exemplary sustained-release enhancing agents include, but are not limited
to hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate
(Poly-HEMA), polyethylene glycol (PEG), glyme and
polyisopropylacrylamide.

[0245] Attaching the amino acid sequence component of the peptides of the
invention to other non-amino acid agents may be by covalent linking, by
non-covalent complexion, for example, by complexion to a hydrophobic
polymer, which can be degraded or cleaved producing a compound capable of
sustained release; by entrapping the amino acid part of the peptide in
liposomes or micelles to produce the final peptide of the invention. The
association may be by the entrapment of the amino acid sequence within
the other component (liposome, micelle) or the impregnation of the amino
acid sequence within a polymer to produce the final peptide of the
invention.

[0246] The compounds described herein may be linear or cyclic (cyclization
may improve stability). Cyclization may take place by any means known in
the art. Where the compound is composed predominantly of amino acids,
cyclization may be via N- to C-terminal, N-terminal to side chain and
N-terminal to backbone, C-terminal to side chain, C-terminal to backbone,
side chain to backbone and side chain to side chain, as well as backbone
to backbone cyclization. Cyclization of the peptide may also take place
through non-amino acid organic moieties comprised in the peptide.

[0247] The peptides of the present invention can be biochemically
synthesized such as by using standard solid phase techniques. These
methods include exclusive solid phase synthesis, partial solid phase
synthesis methods, fragment condensation, classical solution synthesis.
Solid phase peptide synthesis procedures are well known in the art and
further described by John Morrow Stewart and Janis Dillaha Young, Solid
Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

[0248] Synthetic peptides can be purified by preparative high performance
liquid chromatography [Creighton T. (1983) Proteins, structures and
molecular principles. WH Freeman and Co. N.Y.] and the composition of
which can be confirmed via amino acid sequencing.

[0249] Recombinant techniques may also be used to generate the peptides of
the present invention. To produce a peptide of the present invention
using recombinant technology, a polynucleotide encoding the peptide of
the present invention is ligated into a nucleic acid expression vector,
which comprises the polynucleotide sequence under the transcriptional
control of a cis-regulatory sequence (e.g., promoter sequence) suitable
for directing constitutive, tissue specific or inducible transcription of
the polypeptides of the present invention in the host cells.

[0250] In addition to being synthesizable in host cells, the polypeptide
of the present invention can also be synthesized using in vitro
expression systems. These methods are well known in the art and the
components of the system are commercially available.

[0251] According to this aspect of the present invention, the dimeric
peptides comprise anti-allergic activity. Such activities may be assayed
using various known in vitro assay systems and in vivo animal models such
as those described in the Examples section below.

[0252] Since the peptides of the present invention are constrained into
dimers, typically they may be characterized by a well-ordered structure,
such as for example a B sheet structure.

[0253] Since the dimeric peptides of the present invention comprise
anti-allergic properties they may be used to treat allergic conditions.

[0254] Thus, according to another aspect of the present invention there is
provided a method of treating an allergic disorder. The method comprises
administering to a subject in need thereof a therapeutically effective
amount of the peptide compositions of the present invention.

[0255] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical symptoms of
a condition or substantially preventing the appearance of clinical or
aesthetical symptoms of a condition.

[0256] Contemplated subjects that may be treated with the dimeric peptides
of the present invention include mammals, such as human beings.

[0257] Exemplary allergic disorders that may be treated according to this
aspect of the present invention are those mediated by a cell type
selected from the group consisting of mucosal-type mast cells,
serosal-type mast cells and/or basophils, without inducing an
anaphylatoxic effect. In some embodiments, the disorder is an allergic
disorder resulting from an IgE- or IgG-mediated (Type I or Type III)
hypersensitivity and/or FcεRI- or FcγR-induced secretory
response.

[0258] Examples of allergic diseases that can be treated by the dimeric
peptides of the invention include, but are not limited to, allergic
rhinitis, including seasonal rhinitis and sinusitis; pulmonary diseases,
such as bronchial asthma; allergic dermatosis, such as urticaria,
angioedema, eczema, atopic dermatitis, and contact dermatitis; allergic
conjuctivitis; gastrointestinal allergies such as those caused by food or
drugs; cramping; nausea; vomiting; diarrhea; irritable bowel disease; and
ophthalmic allergies such as uveitis; cheilitis; vulvitis; and
anaphylaxis. The present invention is also useful in alleviating or
treating the symptoms induced by exposure to toxins, including bee venom
toxins and the like. In a certain embodiment, the allergic disorder is
asthma.

[0259] For the treatment of hay fever, for example, the peptides may be
formulated in the form of spray, aerosol or drops that can be appropriate
for administration to subjects in need to prevent the development of
allergy in the pollen-season. Moreover, it is well known that the
bronchial mucosal surface is the first contact site for inhaled allergens
and, consequently, the response of mast cells to the inhibitory peptides
of the invention administered as spray may be very effective.

[0261] Further according to an aspect of embodiments of the invention
there is provided a use of dimeric peptides described herein in the
manufacture of a medicament for treating an allergic disorder, as
described herein.

[0262] In any of the methods and uses described herein the dimeric
peptides of the present embodiments may be utilized per se or as part of
a pharmaceutical composition.

[0263] Hence, according to another aspect of embodiments of the invention,
there is provided a pharmaceutical composition which comprises a dimeric
peptide as described herein and a pharmaceutically acceptable carrier.

[0264] The phrase "pharmaceutical composition", as used herein refers to a
preparation of one or more of the active ingredients described herein
with other chemical components such as physiologically suitable carriers
and excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.

[0265] As used herein the term "active ingredient" refers to the dimeric
peptides of the present invention accountable for the intended biological
effect. It will be appreciated that a polynucleotide encoding a peptide
of the present invention may be administered directly into a subject (as
is, or part of a pharmaceutical composition) where it is translated in
the target cells i.e. by gene therapy. Accordingly, the phrase "active
ingredient" also includes such polynucleotides.

[0266] Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier," which may be used interchangeably,
refer to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological activity
and properties of the administered compound. An adjuvant is included
under these phrases.

[0267] Herein, the term "excipient" refers to an inert substance added to
a pharmaceutical composition to further facilitate administration of an
active ingredient. Examples, without limitation, of excipients include
calcium carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0268] Techniques for formulation and administration of drugs may be found
in the latest edition of "Remington's Pharmaceutical Sciences," Mack
Publishing Co., Easton, Pa., which is herein fully incorporated by
reference and are further described herein below.

[0269] The peptides of the present invention as active ingredients are
dissolved, dispersed or admixed in a diluent or excipient that is
pharmaceutically acceptable and compatible with the active ingredient as
is well known. Suitable carriers or excipients are, for example, water,
saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or
the like and combinations thereof. Other suitable carriers are well known
to those in the art. In addition, if desired, the composition can contain
minor amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, stabilizers, binders (e.g., povidone,
gelatin, hydroxypropylmethyl cellulose), lubricants, disintegrants (e.g.,
sodium starch glycollate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose), surface active agents, thickeners,
anti-oxidants, and the like.

[0270] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, sonicating, granulating, grinding,
pulverizing, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.

[0271] Pharmaceutical compositions for use in accordance with the present
invention can be formulated in conventional manner using one or more
physiologically acceptable carriers or excipients comprising auxiliaries,
which facilitate processing of the active ingredients into preparations
which, can be used pharmaceutically. Proper formulation is dependent upon
the route of administration chosen.

[0272] For administration by inhalation, the pharmaceutical compositions
according to the present invention can be delivered in the form of an
aerosol spray presentation from a pressurized pack or a nebulizer with or
without the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In
the case of a pressurized aerosol, the dosage unit may be determined by
providing a valve to deliver a metered amount. Capsules and cartridges
of, e.g., gelatin for use in an inhaler or insufflator can be formulated
containing a powder mix of the peptide and a suitable powder base such as
lactose or starch.

[0273] For administration into eyes, the pharmaceutical compositions can
be delivered as eye drops or eye cream using one or more physiologically
acceptable excipients as known in the art.

[0274] Pharmaceutical compositions, which can be used orally, include
push-fit capsules made of gelatin as well as soft, sealed capsules made
of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit
capsules can contain the active ingredients in admixture with filler such
as lactose, binders such as starches, lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules, the
active compounds can be dissolved or suspended in suitable liquids such
as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. All formulations for oral
administration should be in dosages suitable for the chosen route of
administration. For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.

[0275] Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions can be used which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
titanium dioxide, lacquer solutions and suitable organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different
combinations of active compound doses.

[0276] For injection, the compounds of the invention can be formulated in
aqueous solutions, preferably in physiologically compatible buffers such
as Hank's solution, Ringer's solution, or physiological saline buffer.
For transmucosal administration, penetrants appropriate to the barrier to
be permeated are used in the formulation. Penetrants for example,
polyethylene glycol, are generally known in the art.

[0277] Pharmaceutical compositions for parenteral administration include
aqueous solutions of the active ingredients in water-soluble form.
Additionally, suspensions of the active compounds can be prepared as
appropriate oily injection suspensions. Suitable natural or synthetic
carriers are well known in the art (Pillai et al., Curr. Opin. Chem.
Biol. 5, 447, 2001). Optionally, the suspension can also contain suitable
stabilizers or agents, which increase the solubility of the active
ingredients, to allow for the preparation of highly concentrated
solutions. Alternatively, the active ingredient can be in a powder form
for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free
water, before use.

[0278] The pharmaceutical compositions of the present invention can also
be formulated in rectal compositions such as suppositories or retention
enemas, using, e.g., conventional suppository bases such as cocoa butter
or other glycerides.

[0279] Pharmaceutical compositions suitable for use in context of the
present invention include compositions wherein the active ingredients are
contained in an amount effective to achieve the intended purpose. More
specifically, a "therapeutically effective amount" means an amount of a
compound effective to prevent, delay, alleviate or ameliorate symptoms of
an allergic disease of a subject being treated. Determination of a
therapeutically effective amount is well within the capability of those
skilled in the art.

[0280] Toxicity and therapeutic efficacy of the peptides and analogs,
derivatives, or salts thereof described herein can be determined by
standard pharmaceutical procedures in cell cultures or in experimental
animals, e.g., by determining the IC50 (the concentration which provides
50% inhibition) for a subject peptide. The data obtained from these cell
culture assays and animal studies can be used in formulating a range of
dosage for use in human. The dosage may vary depending upon the dosage
form employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by the
individual physician in view of the patient's condition (e.g. Fingl, et
al., 1975, in The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0281] Depending on the severity of the condition to be treated, dosing
can also be a single administration of a slow release composition, with
course of treatment lasting from several days to several weeks or until
cure is effected or diminution of the disease state is achieved. The
amount of a composition to be administered will, of course, be dependent
on the immune status and health of the subject being treated, the
severity of the disease or condition, the manner of administration, and
other relevant factors.

[0282] The pharmaceutical composition may, if desired, be presented in a
pack or dispenser device, such as an FDA (the U.S. Food and Drug
Administration) approved kit, which may contain one or more unit dosage
forms containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as, but not limited to, a blister
pack or a pressurized container (for inhalation). The pack or dispenser
device may be accompanied by instructions for administration. The pack or
dispenser may also be accompanied by a notice associated with the
container in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals, which notice is reflective
of approval by the agency of the form of the compositions for human or
veterinary administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription drugs
or of an approved product insert. Compositions comprising a peptide
composition of the invention formulated in a compatible pharmaceutical
carrier may also be prepared, placed in an appropriate container, and
labeled for treatment of an indicated condition, as detailed herein.

[0283] Thus, according to an embodiment of the present invention, there is
provided an article of manufacture which comprises the pharmaceutical
composition, as described herein, packaged in a packaging material and
identified in print, in or on the packaging material, for use in the
treatment of an allergic disorder, as described herein. The formulations
and administration methods are intended to be illustrative and not
limiting. It will be appreciated that, using the teaching provided
herein, other suitable formulations and modes of administration can be
readily devised.

[0284] It is to be appreciated that peptides are typically less suitable
for oral administration due to susceptibility to digestion by gastric
acids or intestinal enzymes, however the compositions of the present
invention can be administered orally. The pharmaceutical composition of
the present invention can also be administered by any suitable means,
such as topically, intranasally, subcutaneously, intramuscularly,
intravenously, intra-arterially, intraarticularly, intralesionally,
parenterally or into the eyes. Administration by inhalation is
encompassed in the scope of the present invention.

[0285] The dosage of the composition can be administered to the subject in
multiple administrations in the course of the treatment period in which a
portion of the dosage is administered at each administration.

[0286] The peptides of the present invention may be administered as a
monotherapy, or in combination with other therapeutic agents, such as,
for example, anti-inflammatory agents or steroids. Combination therapies
can involve the administration of the pharmaceuticals as a single dosage
form or as multiple dosage forms administered at the same time or at
different times.

[0290] The term "consisting essentially of" means that the composition,
method or structure may include additional ingredients, steps and/or
parts, but only if the additional ingredients, steps and/or parts do not
materially alter the basic and novel characteristics of the claimed
composition, method or structure.

[0291] The word "exemplary" is used herein to mean "serving as an example,
instance or illustration". Any embodiment described as "exemplary" is not
necessarily to be construed as preferred or advantageous over other
embodiments and/or to exclude the incorporation of features from other
embodiments.

[0292] The word "optionally" is used herein to mean "is provided in some
embodiments and not provided in other embodiments". Any particular
embodiment of the invention may include a plurality of "optional"
features unless such features conflict.

[0293] As used herein, the singular form "a", an and "the" include plural
references unless the context clearly dictates otherwise. For example,
the term "a compound" or "at least one compound" may include a plurality
of compounds, including mixtures thereof.

[0294] Throughout this application, various embodiments of this invention
may be presented in a range format. It should be understood that the
description in range format is merely for convenience and brevity and
should not be construed as an inflexible limitation on the scope of the
invention. Accordingly, the description of a range should be considered
to have specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example, description
of a range such as from 1 to 6 should be considered to have specifically
disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2
to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within
that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of
the breadth of the range.

[0295] Whenever a numerical range is indicated herein, it is meant to
include any cited numeral (fractional or integral) within the indicated
range. The phrases "ranging/ranges between" a first indicate number and a
second indicate number and "ranging/ranges from" a first indicate number
"to" a second indicate number are used herein interchangeably and are
meant to include the first and second indicated numbers and all the
fractional and integral numerals therebetween.

[0296] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including, but
not limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means, techniques and
procedures by practitioners of the chemical, pharmacological, biological,
biochemical and medical arts.

[0297] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable subcombination or as suitable in any other described
embodiment of the invention. Certain features described in the context of
various embodiments are not to be considered essential features of those
embodiments, unless the embodiment is inoperative without those elements.

[0298] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below find
experimental support in the following examples.

EXAMPLES

[0299] Reference is now made to the following examples, which together
with the above descriptions illustrate some embodiments of the invention
in a non limiting fashion.

Example 1

Peptide Syntheses and characterization

Peptide Synthesis:

[0300] Peptides were synthesized by the solid phase technique utilizing
`Boc Chemistry` (Merrifield et al., Biochem. 14: 1385-1390, 1964).
Peptides were dissolved in DMSO and stock solutions at a concentration of
20-25 mg/ml were kept at +4° C. In addition similar stock
solutions were also prepared in deionized water. The final concentration
of the peptides in the assays ranged from 25 μM to 200 μM.

[0301] The following monomeric peptides were prepared:

[0302] TV5501 (C3a9): DCCNYITR (denoted C3a9) (SEQ ID NO:3)

[0303] TV5508 (Monomer A): DCSNYITR (a modification of C3a9 as described
herein, in which the second cysteine is modified by serine) (SEQ ID NO:4)

[0304] TV5513 (Monomer B): DSCNYITR (a modification of C3a9 as described
herein, in which the first cysteine is modified by serine) (SEQ ID NO:5)

[0305] To generate dimeric forms of the peptides DSCNYITR and DCSNYITR,
peptides were dissolved in DMSO and water at a required concentration,
(e.g., 10-20 mg/ml), a 1% iodine solution is used as oxidant and added
step-wise, titrating the free thiolate groups. The progress of the
reaction was monitored by HPLC. After the reaction was completed, the
crude reaction mixtures were purified by preparative HPLC. The structures
were verified by MS.

The following dimeric peptides were synthesized:

##STR00007##

Characterization:

[0306] Water Solubility:

[0307] Water solubility was measured by weighing out specific amounts of
the peptides and monitoring complete dissolution by the transparency of
the solutions. The obtained data is presented in Table 3 below. The
values represent relative solubility in deionized water whereby a highest
solubility, of 5 mg/ml, has a value of 9 and all other values are with
reference thereto.

[0308] Circular Dichroism (CD):

[0309] In order to assess possible structural features that characterize
the active peptides, CD measurement were carried out. The CD measurements
were performed on an Applied Photophysics spetrometer in 1 mm light path
cell, at room temperature (about 25° C.). The peptides stock
solutions were usually of 5 mg/ml in water and the measurements were done
at a 100 μg/ml dilution in PBS.

[0310] The results are presented in FIGS. 1A-D and further in Table 3
below, and indicate that dimer A, dimer B and monomer B all exhibit the
indicated amounts of beta-like structure.

[0313] To study the effect of the peptides on antigen-induced response,
RBL-2H3 cells were pre-incubated with various concentrations of the
peptides for 5 minutes at room temperature before the antigen was added
at the pre-determined suboptimal dose. The peptides were present
throughout the assay. After 1 hour incubation at 37 ° C., 25 μl
of the cell supernatants were removed and incubated with 50 μl of
β-hexosaminidase substrate solution (1.3 mg/ml
p-nitrophenyl-N-acetyl-β-D-glucosamine in 0.1 M sodium citrate, pH
4.5) at 37° C. for 45 minutes. The reaction was terminated by the
addition of 150 μl of 0.2 M glycine, pH 10.7, and the optical density
of the samples was measured at 405 nm. The total enzyme content of the
cells/well and the spontaneous enzyme release of the cells/well was
measured in each experiment and performed in triplicates.

[0314] The total enzyme content of the cells/well (following lysis by
triton-x-100) and the spontaneous enzyme release of the cells/well was
measured in each experiment. All experiments were performed in triplicate
and the results were calculated as net secreted enzyme activity (as
fraction or percentage of total cell-content) in response to the
stimulus.

[0315] Activation of Human Basophils:

[0316] The FLOW-CAST Basophil Activation Test, Flow Cytometry Kit
(Buhlmann) was used for these experiments. The suboptimal stimulant,
an-FcεRI specific mAb (α-chain specific) concentration was
first established. To this end, leucocytes of freshly drawn blood were
treated with various dilutions of the FcεRI specific mAb. In most
of the cases a 40× dilution was used as a suboptimal dose. Cells
were incubated with either 100 or 200 μM peptide for 5 minutes at room
temperature followed by the addition of the FcεRI-specific mAb.
After incubation at 37° C. for 40 minutes, double fluorescent
labeling was performed using PE-labeled anti-CD63 and FITC-labeled
anti-IgE (all supplied in the FLOW-CAST Basophil Activation Test, Flow
Cytometry Kit). After incubation of the samples at 4° C. for 30
minutes, red blood cells were lysed and FACS analysis was performed
assessing CD63 expression levels on the IgE.sup.+ cells.

Results

[0317] As shown in FIGS. 2A-F and FIG. 5, the dimeric peptide 5508 (Dimer
A) exerted similar or higher inhibition as compared with the peptide
monomer designated C3a9.

[0318] As illustrated in FIG. 3 and FIG. 6, exemplary peptide dimers of
the present invention showed enhanced inhibition of CD63 expression as
compared to their monomer counterparts.

Example 3

Effect of Dimeric Peptides on Passive Systemic Anaphylaxis

Materials and Methods

[0319] Passive Systemic Anaphylaxis:

[0320] This assay was performed as reported by J. N. Wu et al., (Journal
of Immunology, 2004, 172: 6768-6774). In brief, anesthetized C57 Black
mice (4-5/group) were injected with the monoclonal IgE class DNP specific
antibody (A2 IgE) by retroorbital injection. A day later, a peptide
solution was dripped into the nose of the mice (20 μl of a 500 μm
solution). Ten minutes later, animals were challenged with antigen
(DNP-BSA11) and 5 minutes later blood was taken for determination of
histamine concentration by the Immunotech Histamine-kit.

Results

[0321] FIGS. 4 and 7 show the extent of inhibition of increase in blood
histamine levels in the peptide-pretreated mice following
antigen-challenge. As shown in FIGS. 4 and 7 monomeric peptides as well
as dimeric peptides showed similar inhibition of histamine release,
although dimeric peptide 5513 was found to be more effective.

[0323] The comparative presentation of the data obtained clearly shows (i)
a good correlation between circular dichroism and inhibition activity;
(ii) improved water solubility and inhibition activity of the dimers
formed from monomeric peptides which include a single cysteine residue
(monomeric peptides derived from C3a9 in which one Cys was replaced by
Ser) as compared to the dimers formed from monomeric peptides which
include 2 Cys residues (as in C3a9), and which were not formed by
directing the position of formed disulfide bridge; and (iii) improved
inhibition activity of the dimers formed from monomeric peptides as
compared to the monomeric peptides.

[0324] Overall, these results indicate superior properties and activity of
dimeric peptides according to embodiments of the invention.

[0325] Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in the
art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad scope
of the appended claims.

[0326] All publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by reference into
the specification, to the same extent as if each individual publication,
patent or patent application was specifically and individually indicated
to be incorporated herein by reference. In addition, citation or
identification of any reference in this application shall not be
construed as an admission that such reference is available as prior art
to the present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.